1. Field of the Invention
Certain embodiments of the invention relate to methods for determining the activation state of a signal transduction pathway signaling protein. In at least some embodiments, methods are provided for monitoring the efficacy of a signal transduction inhibitor in a patient. The present invention further provides highly sensitive assays useful in patient populations in which obtaining a large cellular sample is difficult, for example, neonates. Certain embodiments of the invention relate to methods of identifying novel signal transduction pathway protein inhibitors. Certain embodiments of the invention relate to methods for detecting sepsis in a patient sample. Kits for practicing the methods of the invention are also provided.
2. Background
Many diseases are characterized by disruptions in cellular signaling pathways that lead to pathologies including uncontrolled growth and proliferation of cancerous cells, as well as aberrant inflammation processes. Such defects can include changes in the activity of lipid kinases, a class of enzymes that catalyze the transfer of phosphate groups to lipids. These phosphorylated lipids, in turn, recruit downstream proteins that propagate the signals originating from upstream signaling mediators, such as receptor tyrosine kinases and antigen receptors. For example, the protein kinase Akt is recruited by phospholipids to the plasma membrane where it is activated. Once activated, Akt plays a pivotal role in survival both of normal and cancerous tissues.
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that play pivotal roles in signaling pathways downstream from multiple cell surface receptors, controlling growth, proliferation, and cell survival. Active PI3Ks consist of two subunits: a regulatory subunit with a molecular weight of either 85 or 55 kD (p85 or p55), and a catalytic subunit of molecular weight 110 kD (p110). While the regulatory subunits are critical to the function of PI3K, these regulatory subunits also transmit signals independently of PI3-kinase (Ueki et al., J. Biol. Chem. November 28; 278(48): 48453-66 (2003)). It has recently been demonstrated that p85-alpha can induce apoptosis via the inducible transcription factor NFAT3 independent of the PI3K signaling pathway (Song et al., Mol. Cell. Biol. 27: 2713-2731 (2007)).
The PI3K pathway is implicated in various human diseases including diabetes, heart failure, and many cancers (see e.g., Kim et al., Curr. Opin. Investig. Drugs. December; 6(12): 1250-8 (2005)) including colorectal cancer, acute myeloid leukemia, breast cancer, gliomas, and ovarian cancer. Inhibitors of PI3K are being studied as potential therapeutics in a variety of diseases including cancer, heart failure and autoimmune/inflammatory disorders.
The mitogen activated protein kinase (MAPK) signal transduction pathway is also involved in cell proliferation and differentiation. This pathway plays a role in regulating oocyte meiotic maturation (Moriguchi et al., Adv. Pharmacol. 36:121-137 (1996); Murakami et al., Methods in Enzymology 283:584-600 (Dunphy, ed., 1997); Matten et al., Seminars in Dev. Biol. 5:173-181 (1994)).
The MAPK pathway is also involved in the regulation of cell growth, survival, and differentiation (Lewis et al., supra). Furthermore, activated MAPK and/or elevated level of MAPK expression have been detected in a variety of human tumors (Hoshino, R. et al., Oncogene 18:813-822 (1999); Salh, B. et al., Anticancer Res. 19:741-48 (1999); Sivaraman, V. S. et al., J. Clin. Invest. 99:1478-483 (1997); Mandell, J. W et al., Am. J. Pathol. 153:1411-23 (1998); Licato, L. L. et al. Digestive Diseases and Sciences 43, 1454-1464 (1998)) and may be associated with invasive, metastatic and angiogenic activities of tumor cells. Thus, inappropriate activation of the MAPK pathway is an essential feature common to many types of tumors. For this reason, participants in this signaling pathway, such as MEK, are potential targets for cancer therapy.
Several groups have developed assays to monitor inhibitors of various signal transduction pathways. One assay, which detects the activity of inhibitors of the PI3K>Akt pathway, uses peripheral blood platelets (Bowers et al., Mol. Cancer Ther. 6:2600-2607 (2007)). However, this assay only measures the impact of inhibition of this pathway (inhibition of platelet activation), and does not directly monitor phosphorylation (activation) of the target signaling proteins. In addition, technical difficulties make measurement of this pathway using peripheral blood platelets challenging.
Similarly, West et al. (J. Trauma Injury, Inf. and Crit Care 62:805-811 (2008)) reported a flow cytometry based assay to detect sepsis, using the lack of peripheral blood monocyte activation (measured by phosphorylation of p38 and ERK) following in vitro exposure to lipopolysacharide (LPS). However, this assay did not confirm specificity, and did not note the early ERK activation or the secondary activation of all three MAPK pathways. This assay was also susceptible to unacceptably high background levels for a clinically-relevant assay. For example, from their results, the authors conclude that in normal controls, the percent of monocytes expressing phospho-ERK increases from 35% without LPS stimulation, to 58% positive after LPS treatment in vitro (measured at one 15 min time point after LPS addition). In contrast, presumably septic patients showed 25% P-ERK monocytes before LPS addition, which increased less than 10% following LPS addition in vitro.